Electron transfer function versus oxygen delivery: a comparative study for several hexacoordinated globins across the animal kingdom

Caenorhabditis elegans globin GLB-26 (expressed from gene T22C1.2) has been studied in comparison with human neuroglobin (Ngb) and cytoglobin (Cygb) for its electron transfer properties. GLB-26 exhibits no reversible binding for O(2) and a relatively low CO affinity compared to myoglobin-like globins. These differences arise from its mechanism of gaseous ligand binding since the heme iron of GLB-26 is strongly hexacoordinated in the absence of external ligands; the replacement of this internal ligand, probably the E7 distal histidine, is required before binding of CO or O(2) as for Ngb and Cygb. Interestingly the ferrous bis-histidyl GLB-26 and Ngb, another strongly hexacoordinated globin, can transfer an electron to cytochrome c (Cyt-c) at a high bimolecular rate, comparable to those of inter-protein electron transfer in mitochondria. In addition, GLB-26 displays an unexpectedly rapid oxidation of the ferrous His-Fe-His complex without O(2) actually binding to the iron atom, since the heme is oxidized by O(2) faster than the time for distal histidine dissociation. These efficient mechanisms for electron transfer could indicate a family of hexacoordinated globin which are functionally different from that of pentacoordinated globins.     

A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum: MOLECULAR MODELING AND FUNCTIONAL PROPERTIES*

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O₂ with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.     

An N-Myristoylated Globin with a Redox-Sensing Function That Regulates the Defecation Cycle in Caenorhabditis elegans

Globins occur in all kingdoms of life where they fulfill a wide variety of functions. In the past they used to be primarily characterized as oxygen transport/storage proteins, but since the discovery of new members of the globin family like neuroglobin and cytoglobin, more diverse and complex functions have been assigned to this heterogeneous family. Here we propose a function for a membrane-bound globin of C. elegans, GLB-26. This globin was predicted to be myristoylated at its N-terminus, a post-translational modification only recently described in the globin family. In vivo, this globin is found in the membrane of the head mesodermal cell and in the tail stomato-intestinal and anal depressor muscle cells. Since GLB-26 is almost directly oxidized when exposed to oxygen, we postulate a possible function as electron transfer protein. Phenotypical studies show that GLB-26 takes part in regulating the length of the defecation cycle in C. elegans under oxidative stress conditions.     

Unexpected intron location in non-vertebrate globin genes.

The Caenorhabditis elegans and Artemia T4 globin sequences are highly homologous with other invertebrate globins. The intron/exon patterns of their genes display a single intron in the E and G helices respectively. Precoding introns in multirepeat globins are inserted in homologous positions. Comparison of the intron/exon patterns in the known globin gene sequences demonstrates that they are more diverse than first expected but nevertheless can be derived from an ancestral pattern having 3 introns and 4 exons.     

Genome-wide analysis of the globin gene family of C. elegans

The aim of our study was to annotate sequences for 35 putative globins from the nematode Caenorhabditis elegans. All these proteins are expressed, but seven of these differ from the gene predictions in Wormbase. The entire polypeptide sequences for 31 genes and the core globin domain of four proteins were confirmed or corrected. All core globin domains were aligned manually following a procedure that was designed to fit the putative sequences to the crystal structure based alignment of 56 known globin crystal structures. Neighbor-joining analysis of the resulting alignment showed that the majority of these globins are very divergent from each other, possibly suggesting a long evolutionary divergence. The surprisingly high number and low sequence conservation of putative globins in this small organism urges a detailed functional analysis.     

A model of globin evolution

Putative globins have been identified in 426 bacterial, 32 Archaeal and 67 eukaryote genomes. Among these sequences are the hitherto unsuspected presence of single domain sensor globins within Bacteria, Fungi, and a Euryarchaeote. Bayesian phylogenetic trees suggest that their occurrence in the latter two groups could be the result of lateral gene transfer from Bacteria. Iterated psiblast searches based on groups of globin sequences indicate that bacterial flavohemoglobins are closer to metazoan globins than to the other two lineages, the 2-over-2 globins and the globin-coupled sensors. Since Bacteria is the only kingdom to have all the subgroups of the three globin lineages, we propose a working model of globin evolution based on the assumption that all three lineages originated and evolved only in Bacteria. Although the 2-over-2 globins and the globin-coupled sensors recognize flavohemoglobins, there is little recognition between them. Thus, in the first stage of globin evolution, we favor a flavohemoglobin-like single domain protein as the ancestral globin. The next stage comprised the splitting off to single domain 2-over-2 and sensor-like globins, followed by the covalent addition of C-terminal domains resulting in the chimeric flavohemoglobins and globin-coupled sensors. The last stage encompassed the lateral gene transfers of some members of the three globin lineages to specific groups of Archaea and Eukaryotes.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

A phylogenomic profile of globins

Background

Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold.

Results

A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma.

Conclusion

Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.     

Globin gene family evolution and functional diversification in annelids

Globins are the most common type of oxygen-binding protein in annelids. In this paper, we show that circulating intracellular globin (Alvinella pompejana and Glycera dibranchiata), noncirculating intracellular globin (Arenicola marina myoglobin) and extracellular globin from various annelids share a similar gene structure, with two conserved introns at canonical positions B12.2 and G7.0. Despite sequence divergence between intracellular and extracellular globins, these data strongly suggest that these three globin types are derived from a common ancestral globin-like gene and evolved by duplication events leading to diversification of globin types and derived functions. A phylogenetic analysis shows a distinct evolutionary history of annelid extracellular hemoglobins with respect to intracellular annelid hemoglobins and mollusc and arthropod extracellular hemoglobins. In addition, dehaloperoxidase (DHP) from the annelid, Amphitrite ornata, surprisingly exhibits close phylogenetic relationships to some annelid intracellular globins. We have characterized the gene structure of A. ornata DHP to confirm assumptions about its homology with globins. It appears that it has the same intron position as in globin genes, suggesting a common ancestry with globins. In A. ornata, DHP may be a derived globin with an unusual enzymatic function.     

Genetic variation in mouse beta globin cysteine content modifies glutathione metabolism: implications for the use of mouse models

Allelic variation in the mouse beta globin gene complex (Hbb) produces structurally different beta globins in different mouse strains. Like humans, mice with HbbS alleles produce a single beta globin with one reactive cysteine (beta Cys93). In contrast, mice with HbbD alleles produce two structurally different beta globins, each containing an additional cysteine (beta Cys13). beta Cys93 forms mixed disulfides with glutathione and plays a pivotal role in the activities of hemoglobin, glutathione, and nitric oxide. Similar roles for mouse beta Cys13 have not been described. We used capillary electrophoresis to compare reduced glutathione (GSH), glutathione disulfide (GSSG), and S-glutathionyl hemoglobin levels in erythrocytes from inbred C57BL/6J (homozygous HbbS/S) and 129S1/SvImJ (homozygous HbbD/D) mice and their homozygous and heterozygous B6129S/F2J hybrid offspring. S-glutathionyl hemoglobin was nearly undetectable in inbred or hybrid mice with only monocysteinyl beta globins (HbbS/S) but represented up to 10% of total hemoglobin in mice with polycysteinyl beta globins (HbbS/D or HbbD/D). The stepwise increase in beta globin sulfhydryl group concentration in HbbS/S, HbbS/D, and HbbD/D F2 mice was associated with increasing hemoglobin-bound glutathione and decreasing free glutathione (GSH + GSSG) concentrations. Total erythrocyte glutathione (GSH + GSSG + hemoglobin-bound) was not significantly different between groups. In vitro studies showed that beta Cys13 in mouse HbbD beta globins was more susceptible to disulfide exchange with GSSG than beta Cys93. We conclude that reactive beta globin sulfhydryl group concentration is genetically determined in mice, and that polycysteinyl beta globins markedly influence intraerythrocyte glutathione distribution between free and hemoglobin-bound compartments. Although Hbb heterozygosity and polycysteinyl beta globins are common in wild mouse populations, all common human beta globins contain only one reactive cysteine, and homozygosity is the norm. These fundamental differences in mouse and human beta globin genetics have important implications for the study of mouse biology and for the use of some mouse strains as models for humans.     

Genomic organization of zebra finch alpha and beta globin genes and their expression in primitive and definitive blood in comparison with globins in chicken

How alpha and beta globin genes are organized and expressed in amniotes is of interest to researchers in a wide variety of fields. Data regarding this from avian species have been scarce. Using genomic and proteomic approaches, we present here our analysis of alpha and beta globins of zebra finch, a passerine bird. We show that finch alpha globin gene cluster has three genes (alphas 1–3), each orthologous to its chicken counterpart. Finch beta globin gene cluster has three genes (betas 1–3), with an additional pseudogene at the 3′ end. Finch beta3 is orthologous to chicken betaA, but the orthology of beta1 and beta2 to chicken counterparts is less clear. All six finch globins are confirmed to encode functional proteins. Gene expression in both globin gene clusters is regulated developmentally. Adult finch blood has a globin profile similar to that of adult chicken, with high levels of beta3 and alpha3 and moderate levels of alpha2. Finch embryonic primitive blood exhibits a globin profile very different from that of equivalent stage chick embryos, with all six globins expressed at high levels. Overall, our data provide a valuable resource for future studies in avian globin gene evolution and globin switching during erythropoietic development.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Variation of folded polypeptide surface area with probe size

Three types of polypeptide surface area (contact, accessible, and molecular) have been studied as a function of the radius of a probe sphere used to map the surface. The surfaces are: (1) three alpha-helices, the H-helix of myoglobin, the E-helix of leghemoglobin, and an artificial polyalanine helix, each with 26 residues; (2) two globins, myoglobin and leghemoglobin, each with 153 residues; and (3) a two-center model system for which the three types of surface area have been calculated analytically. The two globin helices have almost identical surface areas as a function of probe size as do the two globins. The polyalanine helix surface area is smaller but similar in shape to the globin helix areas. All three helix contact areas tend to the same limit as the probe size increases, and the globin contact areas behave similarly. Fractal dimensions were calculated for the helix and globin contact and molecular surfaces. All fractal dimensions showed strong dependence on probe size. The contact fractal dimension peaks at larger values for both the helices and globins. Most residues do not make contact with large probes (15 A).     

Sequence and evolution of the gene for the monomeric globin I and its linkage to genes coding for dimeric globins in the insect Chironomus thummi

We isolated genomic clones containing sequences encoding globins I and IA from a Chironomus thummi thummi genomic library. Three clones contain globin IA (ctt-1A) genes, while one contains a globin I (ctt-1) gene. The coding regions of the four genes are identical except for the single base substitution accounting for the globin I/IA polymorphism. The noncoding DNA flanking the coding region is more than 98% similar, confirming a previous hypothesis that the globin ctt-1 and ctt-1A genes are alleles. Hemoglobins I and IA are monomeric in the insect hemolymph. Earlier in situ hybridization studies suggested that monomeric and dimeric globin genes are clustered at different chromosomal loci. In situ hybridization of ctt-1 DNA to polytene salivary gland chromosomes places the ctt-1 gene on the same band as genes for the dimeric globins II and VIIB, forcing revision of the earlier hypothesis that genes for monomeric and dimeric globin genes are at different loci. The evolution of the ctt-1 and ctt-1A alleles and of the two globin gene loci are discussed. Correspondence to: G. Bergtrom     

Evolution of Orthologous Intronless and Intron-Bearing Globin Genes in Two Insect Species

While globin genes ctt-2β and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2β and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species. Received: 31 December 1996 / Accepted: 16 May 1997